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Internexin Causes Abnormal Neurofilamentous Accumulations

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Internexin Causes Abnormal Neurofilamentous Accumulations The Journal of Neuroscience, April 15, 1999, 19(8):2974–2986 Overexpression of a-Internexin Causes Abnormal Neurofilamentous Accumulations and Motor Coordination Deficits in Transgenic Mice Gee Y. Ching,1 Chung-Liang Chien,2 Roberto Flores,1 and Ronald K. H. Liem1 1Departments of Pathology and Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032, and 2Department of Anatomy, National Taiwan University School of Medicine, Taipei, Taiwan 100, Republic of China a-Internexin is the first neuronal intermediate filament (IF) pro- Behavioral tests demonstrate that these mice have a deficit in tein expressed in postmitotic neurons of the developing ner- motor coordination as early as 3 months of age, consistent with vous system. In the adult, its expression is restricted to mature the morphological neuronal changes. Our data further demon- neurons in the CNS. To study the potential role of a-internexin strate that the neurofilamentous inclusions also lead to progres- in neurodegeneration, we have generated transgenic mice that sive loss of neurons in the aged transgenic mice. The motor overexpress rat a-internexin. The total levels of a-internexin coordination deficit and the loss of neurons are transgene expressed in the hemizygous and homozygous transgenic mice dosage-dependent. These data yield direct evidence that high were ;2 and ;3 times the normal level, respectively. Overex- levels of misaccumulated neuronal IFs lead to neuronal dys- pression of a-internexin resulted in the formation of cerebellar function, progressive neurodegeneration, and ultimate loss of torpedoes as early as 1 month of age. These torpedoes are neurons. Moreover, the degrees of neuronal dysfunction and abnormal swellings of Purkinje cell axons that are usually seen degeneration are proportional to the levels of misaccumulated in neurodegenerative diseases involving the cerebellum. EM neuronal IFs. studies showed accumulations of high levels of IFs and abnor- mal organelles in the torpedoes and soma of Purkinje cells, as Key words: a-internexin; neurofilament; intermediate fila- well as in the large pyramidal neurons of the neocortex and in ment; neurodegeneration; cytoskeleton; transgenic mice; motor the ventral anterior and posteromedial nuclei of the thalamus. deficits a-Internexin is a neuronal intermediate filament (IF) protein that son’s disease (Munoz et al., 1988; Sasaki et al., 1989; Galloway et is expressed earlier and more abundantly than the neurofilament al., 1992; Trojanowski and Lee, 1994). Recent transgenic mouse triplet proteins (NFTPs) throughout the developing CNS (for models have shown that overexpression of human NF-H (Cote et review, see Fliegner and Liem, 1991). As development continues, al., 1993) or mouse NF-L (Xu et al., 1993) results in axonal the levels of a-internexin decrease, whereas those of the NFTPs swellings and perikaryal accumulation of neuronal IFs in motor increase (Kaplan et al., 1990; Fliegner et al., 1994). In adult CNS, neurons, as well as skeletal muscle atrophy. The mice develop a-internexin is colocalized with the NFTPs in most axons, but is signs of muscle weakness and reduced kinetic activity, reminis- present at lower levels in large neurons and is found in cerebellar cent of those found in ALS. Transgenic mice expressing a point granule cells, which lack the NFTPs (Kaplan et al., 1990). mutant of NF-L show neuronal abnormalities resulting in motor a-Internexin can self-assemble and coassemble with each of the neuron death and skeletal muscle atrophy (Lee et al., 1994). NFTPs into filaments, whereas the NFTPs are obligate hetero- NF-H mutations have also been found in sporadic ALS patients polymers (Ching and Liem, 1993, 1998; Lee et al., 1993). These (Figlewicz et al., 1994). a properties suggest that -internexin may play a role in stabilizing Overexpression of wild-type or mutant NFTPs does not always small-diameter axons and act as a scaffold on which the NFTPs result in ALS-like phenotypes. Transgenic mice that express a coassemble during development. NF-H/b-galactosidase fusion protein show perikaryal accumula- Abnormal accumulations of neuronal IFs are pathological hall- tions of IFs in their motor neurons but do not develop motor marks of many neurodegenerative diseases, such as amyotrophic neuron degeneration (Eyer and Peterson, 1994). The animals lateral sclerosis (ALS), Lewy body-type dementias, and Parkin- begin to show tremors and selective Purkinje cell loss only at 1 year of age (Tu et al., 1997). Their Purkinje cells contain Lewy Received Nov. 9, 1998; revised Jan. 28, 1999; accepted Jan. 28, 1999. body-like inclusions. Transgenic mice that express wild-type hu- This work was supported by Grant NS15182 from the National Institutes of man NF-M have normal motor neurons in the spinal cord and Health. C.-L. Chien was supported by Grant NSC 88-2314-B-002-119 from National exhibit perikaryal accumulations of neurofilaments in other neu- Science Council, Taiwan. We thank Dr. Frank Costantini and Ms. Xiao-lin Liang for generation of transgenic mouse founders, Dr. Mary Bach for helpful advice on the rons at 12, but not 3, months of age (Vickers et al., 1994). behavioral experiments, Ms. Beth Rosen and Mr. Reilly Coch for their excellent Surprisingly, transgenic mice that overexpress wild-type mouse technical assistance, Mr. Adam Nguyen and Ms. Allie Lui for assistance in DNA extraction and tissue sectioning, and Drs. Dongming Sun and Conrad Leung for NF-H or NF-M show neither muscle atrophy nor motor neuron their help in photographic printing. loss, despite prominent axonal swellings and perikaryal neurofila- Correspondence should be addressed to Dr. Ronald Liem, Department of Pathol- mentous accumulations in motor neurons (Wong et al., 1995; ogy, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032. Marszalek et al., 1996). Copyright © 1999 Society for Neuroscience 0270-6474/99/192974-13$05.00/0 Thus, overexpressions of different NFTPs have differential Ching et al. • a-Internexin-Overexpressing Mice with Motor Deficits J. Neurosci., April 15, 1999, 19(8):2974–2986 2975 effects on neurons. Because a-internexin differs from the NFTPs precipitated protein obtained after dialysis was solubilized by boiling in assembly properties and expression pattern, its overexpression briefly in 10 mM Tris-HCl, pH 7.5, containing 1% SDS. For immuniza- tion, the purified antigen (100 mg/immunization) was mixed with 0.2 ml may cause a different type of neuropathy and provide additional saline containing 25 mg of monophosphoryl lipid A plus synthetic treha- insights into mechanisms of neuronal dysfunction and neurode- lose dicorynomycolate emulsion (Ribi Immunochem Research), an ad- generation. Our present study shows that subpopulations of CNS juvant, and injected subcutaneously in two sites on Balb/c mice. Booster neurons appear to be relatively more vulnerable to filamentous injections were given every 3 weeks. Two weeks after the last antigen/ misaccumulations induced by a-internexin overexpression. More- adjuvant injection, another boost was given without adjuvant. Three days later, spleen cells were removed from the immunized mice for fusion over, the transgenic mice show a transgene dosage-dependent with NS-1 myeloma cells, and monoclonal antibodies were produced deficit in motor coordination. according to the method of Kohler and Milstein (1976). After immuno- blotting and immunohistochemical screening, two monoclonal antibod- MATERIALS AND METHODS ies, mAba12 and mAba3G10, were isolated. mAba12 recognizes only rat a-internexin, and mAba3G10 reacts with both mouse and rat Generation of transgenic mice. To obtain a full-length rat a-internexin a-internexin. genomic clone, a 6.4 kb BamHI–XhoI fragment and a 14.5 kb XhoI Antibodies. The following antibodies were obtained commercially (Sig- fragment, isolated from the rat a-internexin genomic clones laG-1 and ma, St. Louis, MO): mouse monoclonal antibodies to NF-L, NF-M, and laG-2 (Ching and Liem, 1991), respectively, were ligated to the BamHI– NF-H (clones NR4, NN18, and N52, respectively); SMI31 and SMI36, SalI digested lDashII phage vector (Stratagene, La Jolla, CA). The which detect the phosphorylated epitopes on NF-M and NF-H; SMI32, resulting genomic clone was designated laD-1. The transgene construct which recognizes the dephosphorylated forms of NF-M and NF-H; and pSP72N-aES16K was cloned by ligating the 13 kb EcoRI fragment from tau-2, which detects both phosphorylated and dephosphorylated forms of laD-1 and the 3.5 kb EcoRI–SalI fragment from laG-2 together with the tau. Rabbit polyclonal antibody to calbindin 28 kDa was purchased EcoRI–SalI digested pSP72N plasmid vector, which was modified from (Swant, Bellinzona, Switzerland). Rabbit polyclonal antibody to GFAP pSP72 (Promega, Madison, WI) by changing the EcoRV cloning site to a was previously described (Wang et al., 1984). NotI site. To obtain the transgene fragment for generation of transgenic Western blot analysis. M mice, the 16.5 kb NotI–SalI fragment from pSP72N-aES16K, consisting Mouse tissues were homogenized in 10 m a 9 Tris-HCl, pH 7.5, and 1% SDS, boiled for 5 min, and then centrifuged at of the entire rat -internexin gene with 1.2 kb of its 5 flanking sequence 3 and 3.5 kb of its 39 flanking sequence, was isolated from agarose gels and 13,000 g for 5 min to remove insoluble materials. The resulting purified by glass powder. The resulting 16.5 kb transgene fragment was supernatant contained total proteins extracted from the tissues. Protein further purified by Sephadex G50 spin columns (Boehringer Mannheim, concentrations were determined by Bradford assay (Bradford, 1976). Equal amounts of proteins were electrophoresed in 8 or 10% Indianapolis, IN) and prepared at 3 mg/ml in the 10 mM Tris-HCl, pH polyacrylamide-SDS gels (Laemmli, 1970) and then electrotransferred to 7.4, 10 mM NaCl, and 0.2 mM EDTA buffer. Transgenic mouse founders were generated by microinjecting the transgene fragment into fertilized nitrocellulose filters (Towbin et al., 1979).
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